Field of the Invention
This invention relates to a method that allows for the differentiation of commonly encountered bacterial isolates that contain specific antibiotic-resistance genes from similar bacterial isolates that do not harbor the antibiotic-resistance gene. Additionally, this invention relates to methods that allow for the differentiation between bacterial strains containing different antibiotic-resistance genes. More particularly, the invention relates to a method for identifying and differentiating bacterial strains, including: 1) obtaining a biological sample, such as blood, urine, and/or any other bodily fluids; 2) processing the biological sample in order to concentrate the bacteria in the biological sample; and 3) running the concentrated bacterial sample through an optical analyzer to obtain intrinsic fluorescence data, which can then be used to determine not only the bacterial strain, but also whether or not the bacterial strain contains an antibiotic-resistance gene. Additionally, methods provided herein also allow using the data to differentiate between bacteria that may contain different antibiotic-resistance genes. Overall, methods provided herein focus on the use of intrinsic fluorescence, to: 1) differentiate between different types of bacteria (i.e. differentiate between different bacterial species); 2) differentiate between same bacterial species containing different antibiotic-resistance genes; and 3) differentiate between bacterial species that carry an antibiotic-resistance gene from those bacterial species that do not carry an antibiotic-resistance gene. Additionally, methods provided herein allow for analysis of various types of collected suspect samples.
Antimicrobial (i.e., antibacterial) resistance occurs when a microbe (i.e., bacteria and/or bacterial strain) acquires a genetic mutation, either spontaneously or by gene transfer, rendering it resistant to the effect of one or more anti-bacterial agents, i.e., antibiotics. Drug-resistant organisms may acquire resistance to first-line antibiotics, necessitating the use of a second-line agent to which the microbe is sensitive. In the case of some bacterial strains that have gained resistance to multiple drugs, resistance to second- and even third-line antibiotics is sequentially acquired.
Resistance may take the form of a spontaneous or induced genetic mutation, or the acquisition of resistance genes from other bacterial species by horizontal gene transfer via conjugation, transduction, or transformation. Many antibiotic-resistance genes reside on transmissible plasmids facilitating their transfer. Antibiotic-resistance plasmids frequently contain genes conferring resistance to several different antibiotics.
The increasing rates of antibiotic-resistant bacterial infections seen in clinical practice stems from antibiotic use both within human and veterinary medicine. Any use of antibiotics can increase an evolutionary selective pressure in a population of bacteria, allowing resistant bacteria to thrive and non-resistant bacteria to die off. As resistance to antibiotics becomes more common, a greater need for alternative treatments arise. Antibiotic-resistance poses a grave and growing global problem to public health. With an increasing number of bacterial strains having resistance to antibiotics, individuals who require medicinal help are unable to acquire the proper treatment they require.
Therefore, it is an object of the present invention to provide a quick, rapid method for identifying bacterial strains that contain antibiotic-resistance genes. More so, the identification of what type, or types, of antibiotic resistance the bacteria strain contains is necessary. Identification of bacterial strains containing antibiotic-resistance genes would greatly aid in the development of drug design and treatment regimens.
Description of Related Art
In general, current-day practice for identifying, isolating, and differentiating bacterial strains with and without antibiotic-resistance genes often involves a complex and lengthy process in microbiology labs. In the current processes, biological samples containing bacteria are first accepted into the lab. In one process, the biological samples are then streaked, using a sterilized loop, on agar plates containing a nutritionally-rich medium (for example, lysogeny broth or any other suitable broth). This agar plate contains spots that have been treated with an antibiotic. Once the specimen has been streaked on the plate, the agar plate is placed into a dedicated incubator for a minimum of 12 hours. The agar plates are then periodically checked for bacterial colony growth. As would be appreciated by one of ordinary skill in the art, if the biological sample contains bacteria, then bacterial colony growth is expected on the spots not containing the antibiotic. If the bacteria has not acquired an antibiotic-resistance gene, growth on the spots containing the antibiotic is not expected. However, if the bacterial strain has acquired an antibiotic-resistance gene, colony growth will occur on the spots that have been treated with the antibiotic. See for example, commonly owned U.S. Patent Application Publication No. 2008/0220465.
In another process, biological samples, upon collection, are sorted, labeled, and then inoculated into glass, round-bottom, test-tubes containing blood agar medium, or any other suitable nutritionally-rich growth medium (e.g., lysogeny broth), using a sterilized loop. The specimens are then inserted into a dedicated incubator for a 12- to 24-hour period. The samples are then observed and screened for positive (i.e., containing bacteria) and negative cultures (i.e., not containing bacteria). Samples that appear to contain positive cultures are processed in order to isolate and suspend the bacteria in a biochemical fluid. This process involves suspension, dilution, vortexing, and turbidity measurements resulting in biochemical waste products. The cultures are then subjected to a species identification and antibiotics susceptibility tests, which exposes the bacterial suspensions to multiple reagents. After another 6- to 24-hour incubation period, the findings are interpreted and reported by lab technicians. This entire process generally takes at least 11, or more, steps and at least 50 hours to obtain specimen results and the process is labor intensive.
Other processes to differentiate and identify between bacterial species and/or strains involves various types of nucleic acid sequencing methods. Briefly, DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA. In these methods, once a biological sample is obtained, the bacteria contained in the biological sample needs to first be amplified. In other words, the biological sample is first collected, it is then used to inoculate a suitable bacterial growth medium (e.g., blood growth medium or lysogeny broth). The inoculated sample is then grown at appropriate conditions for 12-24 hours. Upon growth, bacterial cells are pelleted from the culture medium, lysed, and processed to extract the bacterial DNA. Bacterial DNA is then cleaned, purified, and placed in a DNA sequencer. The growth of the bacteria and isolation of the bacterial DNA not only requires reagents but also produces bio-waste material, and is additionally a timely process. Additionally, nucleic sequencing methods require the use of primer sequences. A primer is a strand of short nucleic acid sequences (generally about 10 base pairs) that serves as a starting point for DNA synthesis. It is required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA. By requiring primer sequences, this method additionally requires some minimal knowledge of the type of bacterial strain. Sequencing, as indicated, can additionally be time consuming and expensive.
In general, current-day practice for identifying, isolating, and differentiating bacterial strains with and without antibiotic-resistance genes, in a typical microbiology lab, is laborious and is a time-consuming process.